The production of pathogenic antibodies is the cause of a number of autoimmune diseases. However, the cellular and molecular mechanisms underlying the production of pathogenic autoantibodies are incompletely understood. The overall goal of the continuation of the Program Project in Autoimmunity is to develop a more comprehensive understanding of the mechanisms underlying the generation of autoantibodies. The three projects are focused on potential molecular mechanisms controlling generation of autoantibodies and involve a comprehensive and integrated analysis of a number of hypotheses potentially underlying autoantibody production in patients with autoimmune disease. The first project, of which Dr. Peter Lipsky is the principal investigator, will use a novel technique, PCR amplification of genomic DNA from individual B cells, to analyze the immunoglobulin repertoire in patients with SLE, as well as the immunoglobin genes encoding anti-Ro and anti-dsDNA antibodies. This project will examine a number of hypothesis concerning the possibility that abnormalities in V(D)J recombination, somatic hyper-mutation or clonal selection play a role in the development of autoantibodies. In the second project, Dr. Katheryn Meek will examine the hypothesis that abnormal V(D)J recombination leading to excess numbers of charged amino acids in the CDR3 region may predispose toward the production of autoantibodies. This project will use a number of approaches to probe the molecular regulation of D segment utilization and potential perturbations of this in autoimmune mice and humans. Finally, in the third project, Dr. Betty Diamond will explore the basis of the clinical observation that autoimmune diseases are more frequent in women and exacerbated by estrogen, by evaluating the impact of estrogen on repertoire selection and emergence of autoantibody producing B cells. Each of these three projects will focus on molecular mechanisms controlling repertoire generation and selection and should provide important new insights into regulation of the normal immunoglobin repertoire generation and selection and should provide important new insights into regulation of the normal immunoglobin repertoire as well as perturbations in those control mechanisms that might predispose to the production of autoantibodies in patients with autoimmune disease. It is anticipated that as a result of the proposed work, comprehensive new insights into the molecular mechanisms of autoimmunity will arise that could provide novel approaches to therapeutic interventions in the future.